TY - JOUR

T1 - Derivatives of Bst-like Gss-polymerase with improved processivity and inhibitor tolerance

AU - Oscorbin, Igor P.

AU - Belousova, Ekaterina A.

AU - Boyarskikh, Ulyana A.

AU - Zakabunin, Aleksandr I.

AU - Khrapov, Evgeny A.

AU - Filipenko, Maksim L.

N1 - © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

PY - 2017/9/19

Y1 - 2017/9/19

N2 - At the moment, one of the actual trends in medical diagnostics is a development of methods for practical applications such as point-of-care testing, POCT or research tools, for example, whole genome amplification, WGA. All the techniques are based on using of specific DNA polymerases having strand displacement activity, high synthetic processivity, fidelity and, most significantly, tolerance to contaminants, appearing from analysed biological samples or collected under purification procedures. Here, we have designed a set of fusion enzymes based on catalytic domain of DNA polymerase I from Geobacillus sp. 777 with DNA-binding domain of DNA ligase Pyrococcus abyssi and Sto7d protein from Sulfolobus tokodaii, analogue of Sso7d. Designed chimeric DNA polymerases DBD-Gss, Sto-Gss and Gss-Sto exhibited the same level of thermal stability, thermal transferase activity and fidelity as native Gss; however, the processivity was increased up to 3-fold, leading to about 4-fold of DNA product in WGA which is much more exiting. The attachment of DNAbinding proteins enhanced the inhibitor tolerance of chimeric polymerases in loop-mediated isothermal amplification to several of the most common DNA sample contaminants-urea and whole blood, heparin, ethylenediaminetetraacetic acid, NaCl, ethanol. Therefore, chimeric Bst-like Gss-polymerase will be promising tool for both WGA and POCT due to increased processivity and inhibitor tolerance.

AB - At the moment, one of the actual trends in medical diagnostics is a development of methods for practical applications such as point-of-care testing, POCT or research tools, for example, whole genome amplification, WGA. All the techniques are based on using of specific DNA polymerases having strand displacement activity, high synthetic processivity, fidelity and, most significantly, tolerance to contaminants, appearing from analysed biological samples or collected under purification procedures. Here, we have designed a set of fusion enzymes based on catalytic domain of DNA polymerase I from Geobacillus sp. 777 with DNA-binding domain of DNA ligase Pyrococcus abyssi and Sto7d protein from Sulfolobus tokodaii, analogue of Sso7d. Designed chimeric DNA polymerases DBD-Gss, Sto-Gss and Gss-Sto exhibited the same level of thermal stability, thermal transferase activity and fidelity as native Gss; however, the processivity was increased up to 3-fold, leading to about 4-fold of DNA product in WGA which is much more exiting. The attachment of DNAbinding proteins enhanced the inhibitor tolerance of chimeric polymerases in loop-mediated isothermal amplification to several of the most common DNA sample contaminants-urea and whole blood, heparin, ethylenediaminetetraacetic acid, NaCl, ethanol. Therefore, chimeric Bst-like Gss-polymerase will be promising tool for both WGA and POCT due to increased processivity and inhibitor tolerance.

KW - Catalytic Domain

KW - Cloning, Molecular

KW - DNA/metabolism

KW - DNA Polymerase I/antagonists & inhibitors

KW - Enzyme Inhibitors/pharmacology

KW - Genome, Human

KW - Geobacillus/enzymology

KW - Geobacillus stearothermophilus/enzymology

KW - Humans

KW - Nucleic Acid Amplification Techniques/methods

KW - Protein Engineering/methods

KW - Protein Stability

KW - Pyrococcus abyssi/genetics

KW - Recombinant Fusion Proteins/genetics

KW - Sulfolobus/genetics

UR - http://www.scopus.com/inward/record.url?scp=85031898426&partnerID=8YFLogxK

U2 - 10.1093/nar/gkx645

DO - 10.1093/nar/gkx645

M3 - Article

C2 - 28934494

AN - SCOPUS:85031898426

VL - 45

SP - 9595

EP - 9610

JO - Nucleic Acids Research

JF - Nucleic Acids Research

SN - 0305-1048

IS - 16

ER -